A biochemical and genetic discovery pipeline identifies PLCδ4b as a non-receptor activator of heterotrimeric G proteins

Maziarz, M., Broselid, S., DiGiacomo, V., Park, J.-C., Luebbers, A., Garcia-Navarrete, L., Blanco-Canosa, J. B., Baillie, G. S. and Garcia-Marcos, M. (2018) A biochemical and genetic discovery pipeline identifies PLCδ4b as a non-receptor activator of heterotrimeric G proteins. Journal of Biological Chemistry, 293(44), pp. 16964-16983. (doi: 10.1074/jbc.RA118.003580) (PMID:30194280) (PMCID:PMC6222092)

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Abstract

Recent evidence has revealed that heterotrimeric G proteins can be activated by cytoplasmic proteins that share an evolutionarily conserved sequence called the Gα-binding-and-activating (GBA) motif. This mechanism alternative to canonical activation by GPCRs plays important roles in cell function and its dysregulation is linked to disease such as cancer. Here we describe a discovery pipeline that uses biochemical and genetic approaches to validate GBA candidates identified by sequence similarity. First, putative GBA motifs discovered in bioinformatics searches were synthesized on peptide arrays and probed in batch for Gαi3 binding. Then, cDNAs encoding proteins with Gαi3-binding sequences were expressed in a genetically-modified yeast strain that reports mammalian G protein activity in the absence of GPCRs. The resulting GBA motif candidates were characterized by comparison of their biochemical, structural, and signaling properties with those of all previously described GBA motifs in mammals (GIV/Girdin, DAPLE, Calnuc, and NUCB2). We found that the phospholipase C δ4 (PLCδ4) GBA motif binds G proteins with high affinity, has guanine-nucleotide exchange factor activity in vitro, and activates G-protein signaling in cells, as indicated by bioluminescence resonance energy transfer (BRET)-based biosensors of G-protein activity. Interestingly, the PLCδ4 isoform b, which lacks the domains required for PLC activity, bound and activated G proteins more efficiently than the full-length isoform a, suggesting that PLCδ4b functions as a G-protein regulator rather than as a PLC. In summary, we have identified PLCδ4 as a non-receptor activator of G proteins, and established an experimental pipeline to discover and characterize GBA motif-containing proteins.

Item Type:Articles
Additional Information:This work was supported by NIH grants R01GM108733 and R01GM130120, and American Concer Society grant RSG-13-362-01-TBE (to MG-M) and by Medical Research Council grant MR/M013944/1 (to GSB).
Keywords:G protein-coupled receptor (GPCR), GTPase, Phospholipase C, bioluminescence resonance energy transfer (BRET), guanine nucleotide exchange factor (GEF), heterotrimeric G protein.
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Baillie, Professor George
Authors: Maziarz, M., Broselid, S., DiGiacomo, V., Park, J.-C., Luebbers, A., Garcia-Navarrete, L., Blanco-Canosa, J. B., Baillie, G. S., and Garcia-Marcos, M.
College/School:College of Medical Veterinary and Life Sciences > Institute of Cardiovascular and Medical Sciences
Journal Name:Journal of Biological Chemistry
Publisher:American Society for Biochemistry and Molecular Biology, Inc.
ISSN:0021-9258
ISSN (Online):1083-351X
Published Online:07 September 2018
Copyright Holders:Copyright © 2018 the American Society for Biochemistry and Molecular Biology, Inc.
First Published:First published in Journal of Biological Chemistry 293(44): 16964-16983
Publisher Policy:Reproduced in accordance with the publisher copyright policy

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
691651Pathway-driven identification of therapeutic targets for combating Alzheimer's disease.George BaillieMedical Research Council (MRC)MR/M013944/1RI CARDIOVASCULAR & MEDICAL SCIENCES